Abstract
Proteins, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, to explore the impact of the genome-wide interruption on certain protein, we developed a cell lysate microarray on kilo-conditions (CLICK) with 4837 knockout (YKO) and 322 temperature-sensitive (ts) mutant strains of yeast (Saccharomyces cerevisiae). Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3, which covers most of the known regulators in S. cerevisiae We also noted that several group of proteins are involved in negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.
Highlights
Genetic perturbation has been widely employed to illuminate gene function in biological research
Using antibodies specific for H3K4me3 and H3K36me3, we demonstrate that most of the known regulators for these two histone marks could be discovered in a single round of the cell lysate microarray on kilo-conditions (CLICK) assay
Taking the histone mark as an example, in some cell lysates with positive-regulators of histone marks (RHMs) or negative-RHMs genetic perturbation, the histone mark level could be decreased or increased, respectively. These cell lysates are deposited onto a microarray which will be probed with two different primary antibodies simultaneously: one that targets histones generically and another that is specific for a particular histone mark
Summary
Genetic perturbation has been widely employed to illuminate gene function in biological research. The major performer in cell processes, should be a better readout for genetic perturbation. Genome-wide perturbation method to identify the network of genetic regulators on protein state, including its abundance and post-translational level, are still largely lacking. A method using random mutagenesis in haploid human cells is applied to protein measurements in individual cells (Brockmann et al, 2017). This method relies on fluorescence activated cell sorter (FACS) and deep sequencing which require professional and complicate operation. There is a need to develop a quick and efficient method for globally probing of the genetic regulators and the underlying network on protein state
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